Synthetic RNA polymerase III promoters facilitate high efficiency CRISPR-Cas9 mediated genome editing in Yarrowia lipolytica

Schwartz, C.M. et al. ACS Synthetic Biology (2016)

Yarrowia lipolytica is a versatile oleaginous yeast used to model lipid pathways and  produce polymers commercially. However, its use has been limited due to its reliance on selectable markers during genome engineering.  Researchers from the University of California-Riverside have used CRISPR/Cas9 technology to develop a new modular plasmid named pCRISPRyl that can induce homologous recombination with ~60% efficiency without the use of selectable markers.

Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Richardson, C.D. et al. Nature Biotech (2016)

After DNA cleavage with CRISPR/Cas9, cleaved DNA is repaired by one of two mechanisms.  Non-homologous end joining (NHEJ) is an error-prone repair mechanism that usually results in a non-functional gene product and is commonly used by researchers to knockout targeted genes.  Homology-directed repair (HDR) is the insertion of DNA sequences that have ends homologous to the cleavage sight and can be used for precise sequence replacement/addition to the genome, however the efficiency of HDR is extremely low.  Through the use of in vitro kinetic studies, Richardson et al. determined that Cas9 has a very slow rate of dissociation (~6 hours) with targeted DNA, however the non-target strand is released much faster with Cas9 remaining bound to only the targeted strand.  By rationally designing single-stranded DNA donor templates complementary to the non-target strand Richardson et al. increased the efficiency of HDR in human cell lines up to 60% when using eight wild-type or nickase variants of Cas9, and up to 0.7% when using a catalytically dead Cas9 that binds, but cannot cleave DNA.

Scientists Capture Crispr’s Gene-Cutting in Action

Sarah Zhang,, January 14, 2016.

Despite the importance of Cas9, no one has observed exactly how Cas9 cleaves targeted DNA.  Previous crystal structures place the nuclease cleavage domains too far away for DNA cleavage to occur.  In a recent Science publication ( Jennifer Doudna’s group solved the crystal structure of Cas9 primed to cut targeted DNA.  This crystal structure may allow the engineering of more accurate Cas9 endonucleases.

U.K. researcher details proposal for CRISPR editing of human embryos

Erik Stokstad, Science Magazine, January 13, 2016.

Researchers at the Francis Crick Institute in London have applied for permission to modify a human embryo.  The researchers want to use CRISPR/Cas9 technology to study how a fertilized egg turns into a blastocyst, as well as uterus implantation.  All embryos will come from those left after in vitro fertilization attempts and will be destroyed 7 days after fertilization.

A Scientist’s Contested History of CRISPR

Antonio Regalado, January 19, 2016, MIT Technology Review.

The disclosure in “The Heroes of CRISPR” article recently published in Cell has set off a firestorm of criticism.  The conflict stems from Lander’s position as head of the Broad Institute which is currently in a patent dispute with UC-Berkley over the patents for the CRISPR/Cas9 technology.  Regalado points out that Doudna has also failed to always state conflict of interests.

“Heroes of CRISPR” Disputed

Tracy Vence, January 19th, 2016.  The Scientist Magazine–Disputed/

Several scientists have called the accuracy of some sections of “The Heroes of CRISPR” into question.    Dr. Jennifer Doudna and Dr. George Church both state that there are factual errors in the piece, with Doudna stating in a comment posted on PubMed Commons, “the description of my lab’s research and our interactions with other investigators is factually incorrect, was not checked by the author and was not agreed to by me prior to publication.”

The Heroes of CRISPR

Eric S. Lander, Cell (2016) 164:18-28

Major scientific discoveries rarely, if ever, occur in a single lab at a single moment in time.  Instead they involve many people, institutions, and years.  The discovery of CRISPR is no different with story spanning two decades.  This review by Eric Lander focuses on the main players that worked toward understanding the CRISPR loci while making an argument for funding basic science and the promotion of “hypothesis-free” research based on big data.

Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9.

Korkmaz, G. et al (2016) Nature Biotechnology.

Enhancer elements are genomic sequences that regulate the transcription of distantly located genes by acting as binding sites for transcription factors within the chromatin loops.  Enhancer elements are thought to play a significant role in cancer development by altering gene expression. Study of enhancer elements has been limited due to challenges in modifying enhancer sequences.  CRISPR/Cas9 technology may have overcome this with Korkmaz et al presenting a procedure to rapidly modify enhancer elements to study their downstream effects.  The authors went on to characterize two enhancer elements in the p53 and ERα oncogenes.

A face-lift for biotech rules begins.

Emily Waltz, Nature Biotechnology (2015) 33:1221-1222.

On October 3, 2015 the US government held the first of three public meetings to modernize federal regulations for genetically modified (GM) products.  The last time the regulations were updated was in 1986, and with the advent of new GM technologies such as CRISPR/Cas, the system is in strong need of updating.  Two of the main questions facing US federal agencies are: which agency is responsible for oversight, and should GM designation be based upon the end product or the process used.  Currently gene editing techniques, such as CRISPR, that do not add foreign DNA are not considered GM and thus not subject to the GM regulations.  Whether or not the definition of GM products will be expanded to include technologies such as CRISPR remains to be seen.

Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins

Bondy-Denomy, J. et al.  Nature (2015) 526:136-139.

The CRISPR/Cas gene editing system is based upon an adaptive bacterial immune system against bacteriophages.  To overcome this immune system, phages have evolved proteins that inhibit CRISPR/Cas systems.  The mode of inhibition varies by type of Cas system targeted, but usually involves inhibiting the formation of the DNA-Cas complex.  Anti-Cas proteins might find use in gene editing techniques by modulating the activity of the Cas complex.